【摘要】 为研究新型猪繁殖与呼吸综合征疫苗,采用基因克隆技术将合成的特异性猪繁殖与呼吸综合征病毒(PRRSV)RNA干扰寡核苷酸序列克隆至pAAV Helper-Free System中表达质粒pAAV-U6-IRES-hrGFP的U6启动子下游,将PRRSV的ORF5、ORF6基因克隆至pAAV-U6-IRES-hrGFP的CMV启动子下游,构建重组质粒pAAV-U6-IRES-hrGFP-shRNA-ORF5-ORF6;将该质粒与系统中的控制质粒pAAV-RC、辅助质粒pHelper用磷酸钙法共转染293T细胞,包装得到表达PRRSV shRNA、GP5及M蛋白的重组腺相关病毒rAAV-shRNA-ORF5-ORF6。原病毒液经分离纯化后,用点杂交法测定的重组病毒的物理效价高达1010copies/mL。Western-blot检测该重组病毒感染293T细胞后GP5、M蛋白的表达,并通过PRRSV抑制试验检测rAAV-shRNA-ORF5-ORF6的干扰作用。结果显示,成功构建了重组腺相关病毒rAAV-shRNA-ORF5-ORF6,并能在体外表达GP5和M蛋白;rAAV-shRNA-ORF5-ORF6转导Marc145细胞后能够抑制PRRSV的复制。表明成功构建了重组腺相关病毒rAAV-shRNA-ORF5-ORF6,为新型猪繁殖与呼吸综合征双功能疫苗的研制奠定了基础。更多还原

【Abstract】 In order to develop a novel vaccine against porcine reproductive and respiratory syndrome virus(PRRSV),a short interfering RNA(shRNA) against ORF7 of PRRSV was cloned into the downstream of the U6 promoter of pAAV-U6-IRES-hrGFP,and then ORF5 and ORF6 of PRRSV were cloned into the downstream of the CMV promoter of this expression vector to construct recombinant vector pAAV-U6-IRES-hrGFP-shRNA-ORF5-ORF6.293T cells were co-transfected by pAAV-U6-IRES-hrGFP-shRNA-ORF5-ORF6 coupled with pAAV-RC and pHelper to produce a recombinant adeno-associated virus(AAV) co-expressing GP5 and M proteins as well as a shRNA against ORF7 of PRRSV.The results showed that the recombinant rAAV-shRNA-ORF5-ORF6 was successfully constructed.The physical titer of the recombinant virus was 1010 copies/mL measured by dot-blotting.The co-transfected 293T cells could express the-GP5 and M proteins in vitro confirmed by Western-blot.In addition, rAAV-shRNA-ORF5-ORF6 could suppress the replication of PRRSV in Marc145 cells confirmed by virus inhibition test.The study provides the foundation for the development of novel bi-function vaccines against PRRSV.更多还原